It is proposed to determine the number and location of the H ion extruding sites in the electron transport chain of mitochondria isolated from various lines of tumor cells, in order to determine whether there are any functional differences in this aspect of respiratory-energy transduction between normal and tumor mitochondria. In particular, each of the three energy-conserving segments of the chain will be isolated and tested separately for its H ion/2e- ejection ratio and for the number of electrical charges translocated across the membrane per pair of electrons. Special attention will be placed on site 3, namely the cytochrome oxidase reaction, because of its great importance in the regulation of the rate of respiration and ATP synthesis in intact cells. To this end we will examine not only the H ion stoichiometry but also the oxygen kinetics of cytochrome oxidase, in view of the fact that tumor cells can often survive aerobic conditions considerably better than normal cells. We shall also examine the H ion stoichiometry of mitochondrial F0F1ATPase activity in intact tumor mitochondria to determine the number of H ion that must be moved through the H ion "pore" of the ATPase in order to synthesize one molecule of ATP from ADP and phosphate during oxidative phosphorylation. These measurements will be compared with those carried out on normal liver mitochondria. We also propose to determine the stoichiometric relationships between the ADP3- (out)/ATP4-(in) exchange and its reversal, i.e., the ATP4- (out)/ADP3- (in) exchange, a process that takes place during energy-dependent reversal of electron flow and is the vehicle by which the cytosolic phosphorylation potential is communicated to the redox state of the mitochondrial electron carriers. In this way we hope to probe the dynamics of the regulation of ATP energy production by tumor mitochondria. These data will provide a better basis to understanding our previously reported finding that tumor mitochondria take up Ca2 ion with high avidity and that intramitochondrial Ca2 ion inhibits oxidative phosphorylation in tumor mitochondria severely.